Marine ascidians are an excellent source of natural products (Davidson. S. Chem. Rev. 93, 1771-1791 (1993)), including ˜60 cyclic peptides of the patellamide class, which constitute about 6% of natural products isolated from ascidians (MarinLit). Patellamide-containing ascidians harbor cyanobacterial symbionts, Prochloron spp., that have eluded cultivation. Recently, it was shown that Prochloron didemni is responsible for synthesizing cyclic peptides (Schmidt, E. W. et al. Proc. Nat. Acad. Sci. USA 102, 7315-7320 (2005); Long, P. F., Dunlap, W. C., Battershill, C. N. & Jaspars, M. ChemBioChem 6, 1760-1765 (2005)) that were originally isolated from the host ascidians (Ireland, C. M., Durso, A. R., Newman, R. A. & Hacker, M. P. J. Org. Chem. 47, 1807-1811 (1982); Degnan, B. M. et al. J. Med. Chem. 32, 1349-1354 (1989)). Surprisingly, single point mutations in short cassettes in the biosynthetic gene clusters resulted in a diverse product library (Donia, M. S. et al. Nat. Chem. Biol. 2, 729-735 (2006)). By mimicking this natural evolution, a new cyclic peptide was made using rational genetic engineering (Donia 2006.). A homologous pathway was found in the genome of a free-living cyanobacterium encoding a new natural product, trichamide (FIG. 1, peptide 2) (Sudek, S., Haygood, M. G., Youssef, D. T. & Schmidt, E. W. Appl. Environ. Microbiol. (2006)).
The patellamides (FIG. 1, peptides 3-8) are biosynthesized through a unique ribosomal route (Long et al.) with some similarity to microcin pathways (FIG. 1) (Li, Y.-M., Milne, J. C., Madison, L. L., Kolter, R. & Walsh, C. T. Science 274, 1188-1193 (1996)). The products' amino acid sequence is encoded directly on a precursor peptide, PatE (FIG. 1f) (Long et al). Short cassettes within the precursor peptide gene are hypervariable, resulting in a natural combinatorial library of cyclic peptides (Donia 2006). Outside of these cassettes, all known patellamide pathways are 99% identical to each other over their entire lengths (˜11 kb) (Donia 2006). The following biosynthetic hypothesis was proposed (Long 2005; Sudek 2006). The encoding cassettes are flanked by recognition sequences that recruit enzymes for post-translational modifications. Among those are two proteases, PatA and PatG, which catalyze N—C terminal cyclization. PatD is responsible for heterocyclization of cysteine to thiazoline, which is then oxidized to thiazole by an oxidase domain in PatG. PatF may be involved in serine and threonine heterocyclization to oxazoline, among other possibilities. The proteins PatB and PatC were shown to be nonessential (Donia 2006).
Bacterial secondary metabolites are bioactive small molecules that often find use as pharmaceuticals. (Newman et al. J. Nat. Prod. 66, 1022-1037 (2003)). Numerous studies of secondary metabolite biosynthetic genes have led to an increasing ability to synthesize new small molecules through rational pathway engineering (Floss J. Biotechnol. epub (2006); Walsh, C. T. ChemBioChem, 124-134 (2002)). Much of this capability comes from gene sequence comparison, in which the observation of evolution of these pathways has enabled engineering. Despite the advances, a weakness of this approach is that most described pathways are relatively distantly related, making an analysis of single evolutionary events difficult to discern. This difficulty is compounded by the large number of dedicated enzymatic steps (up to approximately 60 or so) commonly required to synthesize individual secondary metabolites.
Small, cyclic peptides are valuable pharmaceuticals, biotechnological products, and tools for scientific research (Davies, J. S. Amino Acids, Peptides and Proteins 2003, 34, 149-217). Cyclic peptides in general have advantages over their linear relatives in that they sample a more constricted conformational and configurational space. (Payne et al. Curr. Org. Chem. 2002, 6, 1221-1246). Stemming from this basic property, cyclic peptides often have stronger binding constants and favorable pharmacological properties such as resistance to proteases (Fairlie, D. P.; Tyndall, J. D. A.; Reid, R. C.; Wong, A. K.; Abbenante, G.; Scanlon, M. J.; March, D. R.; Bergman, D. A.; Chai, C. L. L.; Burkett, B. A. J. Med. Chem. 2000, 43, 1271-1281). Because of this, numerous investigators have developed means to produce arrays of small, cyclic peptides. Synthetic and enzymatic systems, as well as combinations of the two, have been used successfully on small and medium scale (Davies et al. J. Peptide Sci. 2003, 9, 471-501; Hahn et al. Proc. Nat. Acad. Sci. USA 2004, 101, 15585-15590). At the large scale, peptides in phage-display libraries have been cyclized via disulfide bonds or via semi-synthesis from the same libraries (Kehoe, J. W.; Kay, B. K. Chem. Rev. 2005, 105, 4056-4072; Ho, K. L.; Yusoff, K.; Seow, H. F.; Tan, W. S. J. Med. Virol. 2003, 69, 27-32).
There is a great need for new methods for making cyclic peptides, particularly for the manufacture of synthetic cyclic peptides for clinical investigations and therapeutic use, and for the production of cyclic peptide libraries that can be screened to identify cyclic peptides with a desired activity. What is needed in the art are methods for the in vivo construction of cyclic peptide libraries, as well as the compounds resulting therefrom.